Curative cancer therapy leads to selective cell death of the cancer cells. Unlike necrosis that is associated with an inflammatory tissue response, cell death by apoptosis is silent, and it believed to represent the consequence of a decision made by the cell to commit suicide. We have studied cell death in EBV-immortalized cells. Despite the fact that EBV-immortalized B cells are capable of continuous growth when cultured under optimal culture conditions, they die when incubated at sufficiently low cell densities. Under these culture conditions, cell death is prevented by addition of IL-6 or lactic acid to the culture medium. We have examined the process of death in EBV-immortalized cells cultured at critically low cell densities, and examined whether it occurs by necrosis or apoptosis. Two lines of evidence suggest that death occurs through apoptosis. First, the cellular DNA was found to separate in discrete fragments on agarose gels. Second, the protein synthesis inhibitor cycloheximide inhibited cell death, suggesting that the process involves protein synthesis, a characteristic feature of apoptosis but not necrosis. We tested whether death in factor deprived EBV-immortalized cells occurs at a particular stage of cell cycle. Much of the data supports the view that cell death under these experimental conditions occurs as all stages of cell cycle, without a clear preference for a given stage of cell cycle. In addition, cell cycle analysis of factor deprived cells indicated that the cells fail to growth arrest in GO/1. A number of gene products have been implicated in apoptic death processes, including BCL-2, p53 and c-myc. Levels of expression of p53, BCL-2 and c-myc did not change significantly in EBV-immortalized cells destined to die by apoptosis. The observation that growth-factor deprived EBV-immortalized cells die at all stages of the cell cycle, without undergoing growth arrest, together with the observation that c-myc expression remains unchanged in these cells, suggests that deregulated c-myc expression is an important element of cell death in these cells. In contrast to EBV-immortalized B cells, peripheral blood B cells activated by anti-CD40 anf IL-4, rapidly downregulate expression of c-myc and arrest growth in G0/G1, indicating that these cells can regulate appropriately c-myc expression in response to growth factor deprivation. Thus, EBV-immortalized cells die by apoptosis when deprived of autocrine growth factors and this process is associated with continuing cell cycle progression and insignificant changes in the expression of c-myc, BCL-2 and p53. Thus, EBV-immortalized B cells harbor a deregulated c-myc that is critical to apoptosis. Cherney, B.W., Bhatia K., Tosato G. A role for deregulated c-myc expression in apoptosis of Epstein-Barr virus immortalized B cells. Proc. Natl. Acad. Sci. USA 91: 12967-71, 1994.
